This invention relates to methods of specifically delivering an effector molecule to a tumor cell. In particular this invention relates to chimeric molecules that specifically bind to IL-13 receptors which, when combined with IL-4 receptor blockers, specifically deliver compounds or having a particular activity to tumors overexpressing IL-13 receptors.
In a chimeric molecule, two or more molecules that exist separately in their native state are joined together to form a single entity (molecule) having the desired functionality of all of its constituent molecules. Frequently, one of the constituent molecules of a chimeric molecule is a xe2x80x9ctargeting moleculexe2x80x9d. The targeting molecule is a molecule such as a ligand or an antibody that specifically binds to its corresponding target, for example a receptor on a cell surface. Thus, for example, where the targeting molecule is an antibody, the chimeric molecule will specifically bind (target) cells and tissues bearing the epitope to which the antibody is directed.
Another constituent of the chimeric molecule may be an xe2x80x9ceffector moleculexe2x80x9d. The effector molecule refers to a molecule that is to be specifically transported to the target to which the chimeric molecule is specifically directed. The effector molecule typically has a characteristic activity that is desired to be delivered to the target cell. Effector molecules include cytotoxins, labels, radionuclides, other ligands, antibodies, drugs, prodrugs, liposomes, and the like.
In particular, where the effector component is a cytotoxin, the chimeric molecule may act as a potent cell-killing agent specifically targeting the cytotoxin to cells bearing a particular target molecule. For example, chimeric fusion proteins which include interleukin 4 (IL-4) or transforming growth factor (TGFxcex1) fused to Pseudomonas exotoxin (PE) or interleukin 2 (IL-2) fused to Diphtheria toxin (DT) have been shown to specifically target and kill cancer cells (Pastan et al., Ann. Rev. Biochem., 61: 331-354 (1992)).
Generally, it is desirable to increase specificity and affinity and decrease cross-reactivity of chimeric cytotoxins with targets to be spared in order to increase their efficacy. To the extent a chimeric molecule preferentially selects and binds to its target (e.g. a tumor cell) and not to a non-target (e.g. a healthy cell), side effects of the chimeric molecule will be minimized. Unfortunately, many targets to which chimeric cytotoxins have been directed (e.g. the IL-2 receptor), while showing elevated expression on tumor cells, are also expressed to some extent, and often at significant levels, on healthy cells. Thus, chimeric cytotoxins directed to these targets frequently show adverse side-effects as they bind non-target (e.g., healthy) cells that also express the targeted receptor.
The present invention provides methods and compositions for specifically delivering an effector molecule to a tumor cell. In particular, the present invention provides methods and compositions for specifically targeting target tumor cells while offering reduced targeting of healthy cells than previously known methods and compositions.
The improved specific targeting of this invention is premised, in part, on two discoveries: The first discovery was that tumor cells, especially carcinomas such as renal cell carcinoma, Kaposi""s sarcoma, and brain tumors such as gliomas and medulloblastomas overexpress IL-13 receptors at extremely high levels. The second discovery was that despite the fact that the IL-4 and IL-13 appear to share a common receptor on healthy cells, the receptors are xe2x80x9cdecoupledxe2x80x9d in cancerous cells so that blocking of the IL-4 receptor confers protection of healthy cells without inhibiting the activity of IL-13 receptor directed molecules on cancerous cells. This permits IL-13 receptor-directed chimeric molecules (e.g., IL-13R-cytotoxins) to be administered at higher dosages with fewer adverse side-effects (e.g., IL-13R-cytotoxins administered with an IL-4R blocker will have a higher LD50). In addition, reduction and elimination of any binding between an IL-13R directed chimera and IL-4 receptors will leave greater concentrations of the chimera free in the circulation to bind to IL-13 receptors. These features (among others) coupled with the extremely high level of IL-13 receptor expression on target tumor cells permits the specific delivery of relatively high concentrations of IL-13R-directed chimerics to their IL-13R-bearing target cells.
Thus, in one embodiment, this invention provides a method of specifically delivering an effector molecule to a tumor cell bearing an IL-13 receptor (preferably an IL-13 receptor that is not shared with IL-4). The method involves the steps of: providing a chimeric molecule comprising an effector molecule attached to a targeting molecule that specifically binds to an IL-13 receptor; and contacting the tumor cell with the chimeric molecule in the presence of a blocker of an interleukin-4 receptor (IL-4R). The blocker is preferably present in a concentration sufficient to block binding of the targeting molecule to the IL-4 receptor. The chimeric molecule thus specifically binds to the tumor cell. In a preferred embodiment, particularly where the blocker is a molecule that also occurs endogenously (e.g., IL-4) the blocker is present in a concentration greater than that found in the environment in which the tumor cells and/or healthy (non-tumerous) cells normally occur. Preferred blockers include, but are not limited to an interleukin-4, an interleukin-4 antagonist, and an interleukin-4 receptor binding antibody (anti-IL-4R Ab). Interleukin-4 antogonists are selected whose antagonistic activity is mediated by binding to the IL-4 receptor not to IL-4 itself thus, they act as IL-4 competitors or competitive antagonists. Particularly preferred blockers specifically bind to the 140 kDa subunit of the IL-4 receptor. Preferred blockers include interleukin antagonists such as an interleukin-4 having a mutation in xcex1-Helix D with more preferred blockers including [Y124D]hIL4 and [R121D, Y124D]hIL4.
In preferred chimeric molecules, the targeting molecule is either a ligand, such as IL-13 or an anti-IL-13 receptor antibody. The targeting molecule may be chemically conjugated to the effector molecule, or where both targeting and effector molecules are polypeptides, the targeting molecule may be joined to the effector molecule through one or more peptide bonds thereby forming a fusion protein. Suitable effector molecules include a cytotoxin, a label, a radionuclide, a drug, a prodrug, a liposome, a ligand, and an antibody. In a particularly preferred embodiment, the effector is a cytotoxin, (e.g., Pseudomonas exotoxin, Diphtheria toxin, ricin, abrin, or a cytotoxic prodrug) with Pseudomonas exotoxin or Diptheria toxin (especially truncated forms in which the native binding domain is eliminated) being more preferred and Pseudomonas exotoxin (e.g., PE38QQR, PE4E, etc.) being most preferred. Where the Pseudomonas exotoxin is fused to an IL-13 targeting molecule, preferred fusion proteins include, but are not limited to IL-13-PE38QQR, IL-13-PE4E, cpIL-13-PE38QQR, and cpIL-13-PE4E.
As indicated above, the chimeric molecule is preferably contacted with the tumor cell in the presence of an IL-4 receptor (IL-4R) blocker. Preferred IL-13R-directed chimera/blocker combinations include, but are not limited to IL-13-PE38QQR or IL-13-PE4E and [Y124D]hIL4 or [R121D, Y124D]hIL4.
Preferred targets for the methods of this invention include cells, tissues, or organs that express, more preferably overexpress IL-13 receptors. Particularly preferred targets are tumor cells that overexpress IL-13 receptors. Such tumor cells include, but are not limited to renal cell carcinoma cells, brain tumor cells (e.g., glioma cells, medulloblastoma cells, etc.), and Kaposi""s sarcoma cells.
In another embodiment, this invention provides a method of impairing growth of tumor cells bearing an IL-13 receptor. The method involves contacting the tumor cell with a chimeric molecule comprising a targeting molecule that specifically binds a human IL-13 receptor; and an effector molecule selected from the group consisting of a cytotoxin, a radionuclide, a ligand, an antibody, and a cytotoxic prodrug. The contacting is in the presence of a blocker of an interleukin receptor (IL-4R). The blocker is preferably present in a concentration sufficient to block binding of the targeting molecule to the IL-4 receptor. The chimeric molecule thus specifically binds to the tumor cell. In a preferred embodiment, particularly where the blocker is a molecule that also occurs endogenously (e.g., IL-4) the blocker is present in a concentration greater than that found in the environment in which the tumor cells and/or healthy (non-tumerous) cells normally occur. Any of the IL-4R blockers described herein are suitable. Any of the targeting molecules described herein are suitable targeting molecules in the chimeric molecule and any of the cytotoxic molecules described herein are suitable effector molecules. Preferred IL-13R-directed chimera/blocker combinations include, but are not limited to IL-13-PE38QQR or IL-13-PE4E and [Y124D]hIL4 or [R121D, Y124D]hIL4. Particularly preferred targets are tumor cells that overexpress IL-13 receptors. Such tumor cells include, but are not limited to renal cell carcinoma cells, brain tumor cells (e.g., glioma cells, medulloblastoma cells, etc.), and Kaposi""s sarcoma cells. The tumor cell growth that is inhibited can be tumor cell growth in a human. The contacting step may comprise administering the chimeric molecule to a human intravenously, into a body cavity, or into a lumen or an organ.
In still another embodiment, this invention provides a method of detecting the presence, absence, size, or number of tumor cells. The method involves contacting the tumor cell(s) with a chimeric molecule comprising a targeting molecule that specifically binds a human IL-13 receptor; and a detectable label, and detecting the presence, absence, or quantity of the detectable label. The contacting is in the presence of a blocker of an interleukin receptor (IL-4R) and the blocker is present in a concentration sufficient to block binding of the targeting molecule to an IL-4 receptor. In a preferred embodiment, particularly where the blocker is a molecule that also occurs endogenously (e.g., IL-4) the blocker is present in a concentration greater than that found in the environment in which the tumor cells and/or healthy (non-tumerous) cells normally occur. Suitable blockers and targeting molecules include any of the blockers and targeting described herein. Detectable labels include, but are not limited to those discussed herein.
In still yet another embodiment, this invention provides a pharmacological composition. The composition includes a pharmaceutically acceptable carrier, a chimeric molecule comprising an effector molecule attached to a targeting molecule that specifically binds to an IL-13 receptor, and a blocker of an interleukin receptor (IL-4R). Any of the chimeric molecules, more preferably the cytotoxic chimeras and chimeras in which the effector is a detectable label, and most preferably the cytotoxic chimeras, described herein are suitable. The blocker can include, but is not limited to, any of the blockers described herein.
Definitions
The term xe2x80x9cspecifically deliverxe2x80x9d as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule or expressing that target molecule at low levels. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule. Specific delivery typically results in greater than 2 fold, preferably greater than 5 fold, more preferably greater than 10 fold and most preferably greater than 100 fold increase in amount of delivered molecule (per unit time) to a cell or tissue bearing the target molecule as compared to a cell or tissue lacking the target molecule or marker.
The term xe2x80x9cblockerxe2x80x9d when used with reference to an IL-4 receptor refers to a substance that specifically binds to an IL-4 receptor, or component thereof, and reduces or prevents binding of that receptor by another different substance (e.g., an IL-13 based chimeric molecule). Because the blocker competes with the native ligand (IL-4) for the IL-4 receptor it is also referred to as an IL-4 competitor. Moreover, since most preferred blockers do not activate the IL-4 receptor, the preferred blocker is an xe2x80x9cIL-4 antagonistxe2x80x9d or a xe2x80x9ccompetitive antagonistxe2x80x9d of IL-4. One of skill in the art will appreciate that a blocker, to be effective, need not eliminate all binding to the xe2x80x9cblockedxe2x80x9d receptor but rather a simple reduction in binding of other molecules to the subject receptor is sufficient. The effect of such blocking is to make the receptor, on average, generally less available for binding to moieties other than the blocking agent.
The term xe2x80x9cresiduexe2x80x9d as used herein refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
A xe2x80x9cfusion proteinxe2x80x9d refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone.
A xe2x80x9cspacerxe2x80x9d as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule. A spacer can also be an organic (non-peptide) molecule that serves the same purpose as the peptide spacer.
A xe2x80x9cligandxe2x80x9d, as used herein, refers generally to all molecules capable of reacting with or otherwise recognizing or binding to a receptor on a target cell. Specifically, examples of ligands include, but are not limited to, antibodies, lymphokines, cytokines, receptor proteins such as CD4 and CD8, solubilized receptor proteins such as soluble CD4, hormones, growth factors, and the like which specifically bind desired target cells.
The term xe2x80x9ccpIL-13xe2x80x9d is used to designate a circularly permuted (cp) IL-13. Circular permutation is functionally equivalent to taking a straight-chain molecule, fusing the ends (directly or through a linker) to form a circular molecule, and then cutting the circular molecule at a different location to form a new straight chain molecule having different termini.